There are two objectives of this research proposal: (a) To elucidate the molecular mechanism of ciliary and flagellar motility; and (b) To investigate the processes of assembly of these organelles. The biochemistry and ultrastructure of components isolated from cilia and flagella will be studied with the following specific aims: (1) Sliding has been demonstrated to take place between the central pair microtubule sheath and the radial spokes of doublet microtubules; it is importnat that the mechanism of this sliding be understood. The proteins comprising the spokes and central sheath will be purified and characterized by standard biochemical procedures, and will be localized within the axoneme by extraction/reconstitution experiments, by antibody labelling and by 3-dimensional computer reconstruction of negatively stained axonemes. (2) Methods have been developed in my laboratory for the isolation of the central pair microtubules and their associated sheath components. The ultrastructure, composition and reconstitution of the sheath components will be studied. Structural and chemical interactions between the sheath ans spokes will be studied by recombing soluble and/or reconstituted components of these two structures and monitored by EM, optical diffraction, darkfield microscopy and biochemical methods. (3) Purified spoke and sheath components and dynein 2 will be examined for ATPase and calcium-binding activities and studied to determine how these two properties affect ciliary flagellar motility. (4) Doublet microtubles can be fractionated into resistant ribbons of 3 protofilaments containing 5 proteins plus tubulin. These proteins will be localized and their arrangement on the tubulin lattice determined by optical diffraction analysis of negatively stained material. (5) Purified ribbon proteins will be characterized by amino acid analysis and peptide mapping; their molecular associations in solution and on the ribbons will be determined using bifunctional cross-linking techniques. (6) The function of ribbon proteins will be studied by reassembly experiments in which ribbons and renatured ribbon proteins are recombined with soluble flagellar and brain tubulin.